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Bacteria that infect humans and animals face a hard combat with the host´s immune system and in order to establish infection, pathogenic bacteria has evolved mechanisms to avoid being cleared from the host tissue. Many Gram-negatives carry a Type 3 secretion (T3S) system that is used to deliver effector proteins (toxins) into host cells. The toxins exhibit a broad range of intra cellular effects that has in common that they increase the chances of the bacteria to establish infection, multiply in infected tissue or spread to other tissues or hosts. The object of this study was to analyse the mechanisms behind the T3S effectors delivery into target cells. Two bacterial proteins, LcrV and YopD, which are involved in the translocation of effectors were analyzed by mutagenesis. LcrV was found to affect the efficiency of the translocation, probably by determining the size of the pore in the target cell membrane through which the effectors pass. Truncated variants of the multi-functional YopD revealed that defined regions of the protein were important for pore-formation and translocation. Effectors and translocators were demonstrated to form complexes with free acyl chains (lipids) at the bacterial surface. These complexes –termed Yop-lipid complexes, (YLC)– are released from the surface and can act as discrete units that are able to promote translocation of effectors even when separated from the bacterium from which they originate. These findings shed new light on the process of effector translocation by Yersinia and possibly by other gram-negative bacterial pathogens with a similar T3S setup.

The Type III secretion system (T3SS) is an important virulence mechanism that allows pathogenic bacteria to translocate virulence effectors directly into the cytoplasm of eukaryotic host cells to manipulate the host cells in favor of the pathogen. Enteropathogenic Yersinia pseudotuberculosis use a T3SS to translocate effectors, Yops, that prevent phagocytosis by immune cells, and is largely dependent on it to establish and sustain an infection in the lymphoid tissues of a mammalian host. Translocation into a host cell requires specific translocator proteins, and is tightly controlled from both the bacterial and host cell cytoplasm. We aimed to investigate two of the regulatory elements, YopN and LcrV, to gain more insight into the translocation mechanism. Two separate regulatory complexes regulate expression and secretion of Yops, however, the processes are linked so that expression is induced when secretion is activated. A complex, including YopD, prevents expression of Yops, while YopN-TyeA and LcrG block secretion. LcrV is required to relieve the secretion block, by sequestering LcrG. We verified that LcrG binds to the C-terminal part of LcrV, which is consistent with what has been shown in Y. pestis. In addition to their regulatory roles, both LcrV and YopD are translocators and are assumed to interact at the bacterial surface, where LcrV promotes insertion of YopB and YopD into the host cell membrane. However, here we show that purified YopD failed to interact with LcrV, instead YopD solely interacted with a complex of LcrV-LcrG. This indicates that LcrV and YopD interact in the bacterial cytosol, which may be important for regulation of Yop expression and secretion. The established role of YopN is to block secretion prior to host cell contact. We found that deleting the central region (amino acids 76-181) had no effect on the regulatory role of YopN in expression and secretion of Yops. Interestingly, we found that, even though the YopN∆76-181 mutant secreted the translocators with similar kinetics as the wild type strain, translocation of the effector YopH, into HeLa cells, was significantly reduced. Consequently, the YopN∆76-181 mutant was unable to block phagocytosis, almost to the same level as the ∆lcrV mutant which is completely unable to translocate YopH. Our results indicate that YopN is involved in the translocation step in addition to its role in regulating secretion. Further, we show that the amino terminal of LcrV, in the context of translocation, is involved in the early intracellular targeting of YopH in order to block phagocytosis efficiently and sustain an in vivo infection. LcrV mutants that failed to efficiently target YopH intracellularly were severely attenuated also for in vivo virulence. All together, we show that LcrV and YopN are involved in more steps in the regulation of translocation, than what was known before. Our studies also highlight that early translocation is essential for Yersinia to block phagocytosis, which in the end is essential for in vivo virulence.

Pseudomonas aeruginosa et Yersinia pestis sont responsables d'infections graves chez les individus immunodéprimés et de la peste, respectivement. Leur pathogénicité repose sur de nombreux facteurs de virulence dont le système de sécrétion de type III (SST3) qui a une action prépondérante lors d'infections aiguës. Le SST3 est composé d'une base ancrée dans la double membrane bactérienne, d'une aiguille creuse érigée à la surface et d'un pore de translocation inséré dans la membrane de la cellule hôte permettant à la bactérie d'y injecter des toxines. L'objet de cette thèse est l'étude de l'interactome de l'antigène protecteur V, PcrV chez P. aeruginosa et LcrV chez Y. pestis. Celui-ci est situé au sommet de l'aiguille et est nécessaire au processus de translocation des toxines. L'étude des propriétés biochimiques de la protéine in vitro nous a permis de mettre en évidence sa capacité à former des oligomères présentant une structure en forme d'anneaux. Les multimères ont été observés par chromatographie d'exclusion de taille, gel natif, spectrométrie de masse native et MET. Leur formation est dépendante de la présence de l'hélice α12 C terminale de PcrV et de l'intégrité de ses résidus hydrophobes. Le processus d'assemblage de la protéine est nécessaire à sa fonction in vivo : des mutants qui sont incapables d'oligomériser perdent leur cytotoxicité envers les cellules eucaryotes. <br />Puis, l'interaction directe entre PcrV et la sous unité formant l'aiguille, PscF, a été mise en évidence in vitro par co-purification. De plus, deux mutants ponctuels de PscF dont le phénotype présente un défaut de translocation se sont montrés défectueux pour la liaison avec PcrV. Enfin, l'hélice C terminale de PscF est échangeable avec l'hélice α12 C terminale de PcrV comme l'atteste la capacité de polymérisation d'un hybride créé entre ces deux protéines, suggérant un rôle de celle-ci dans la formation du complexe F-V. L'ensemble de ces études montre que l'assemblage multimérique des antigènes V ainsi que leur position au sommet de l'aiguille sont des éléments essentiels à leur fonction, avec un rôle prépondérant de l'hélice α12 C terminale de PcrV. Ces conclusions pourraient permettre de mieux cibler les développements futurs de nouveaux vaccins ou agents antimicrobiens.